Comparison of serological tests for detection of antibodies to infectious laryngotracheitis virus

Abstract

SUMMARYFour serological tests i.e. ELISA, serum neutralisation (SN), fluorescent antibody (FA), and agar gel immunodiffusion (AGID) were compared for sensitivity using several criteria, for detection and titration of infectious laryngotracheitis (ILT) virus antibodies in chicken sera. In the ELISA test, sera were tested in parallel on virus positive and negative control antigens with results expressed as positive-negative difference. Non-specific binding was not a problem in this test. SN tests were performed in microtitre plates using chick embryo liver cells, while sera for FA tests were titrated on multispot slides on which were fixed ILT virus-infected chick embryo liver cell cultures. The AGID test was the standard test still widely used for serological diagnosis of ILT.The four tests were compared using (1) sera from experimentally inoculated birds bled regularly at intervals from 4 to 35 days post-inoculation, (2) convalescent sera from a natural outbreak of ILT, and (3) serial dilutions of an ILT positive serum. In all experiments the ELISA test was of slightly greater sensitivity than SN and was comparable to the FA test. In the experimentally infected birds ELISA and FA test detected sero-conversion in more birds at 7 days than SN. In tests with the serially diluted hyperimmune ILT serum, ELISA, FA and SN tests were comparable. SN however was the most useful test for quantification of ILT antibodies. ILT-SN titres in birds were never high, the highest titre recorded in experimental birds and in convalescent sera was 1/48. AGID was found to be less sensitive than ELISA, FA or SN test but was considered useful for detection of antibodies on a flock basis, since, with the convalescent sera AGID detected a significant proportion of positives.

Comparison of commercial ELISA test kits from Australia and the USA with the serum neutralization test in cell cultures for the detection of antibodies to the infectious laryngotracheitis virus of chickens

“…This indicates that the sensitivity of the two commercial ILT-ELISAs is superior to the sensitivity of the SNT. This is also con® rmed by other authors (Adair et al, 1985). However, owing to the higher deviations of the ELISA extinctions, it is dif® cult to assign certain extinction values to speci® c dilution steps.…”

“…However, this test is now considered unreliable (Buken, 1982;Fuchs et al, 1985). In contrast, the serum neutralization test (SNT) was shown to be time-consuming, but very reliable, accurate and suitable for quantitative measurements (Adair et al, 1985). It can be performed both in embryonated eggs (Hitchner et al, 1958) and in cell cultures, including the micro-assay SNT (Robertson & Egerton, 1977).…”

“…Furthermore, they used anti-ILT rabbit-antisera in order to avoid the dif® cult production of suf® cient amounts of puri® ed ILT antigen. Adair et al (1985) performed parallel measurements with normal antigen and with negative control antigen, and the result was expressed as the difference between the mean absorbance produced by the serum on the positive and negative antigen wells (absorbance difference). Ohkubo et al (1988) reduced non-speci® c binding by using biotin-linked conjugate and avidin-linked peroxidase.…”

A study of ELISA systems incorporating pooled viral andMycoplasmaantigen preparations for antibody screening of chicken sera

“…Negative antigens have been used by us in some tests (e.g. ILT, AI; Adair et al 1985Adair et al , 1989b where we have experienced higher background absorbance values with some negative field sera on positive virus antigen. Results are expressed as absorbance difference and specificity is consequently increased without loss of sensitivity.…”

“…The methods used for the preparation of ILT, NDV and AI ELISA antigens were described previously (Adair et al, 1985(Adair et al, ,1989a. A/parrot/Ulster/73(H7.Nl) was used for influenza A antigen.…”